The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of two flavin amine oxidases, mouse polyamine oxidase (PAO) and human lysine specific demethylase (LSD1), to determine the mechanisms of amine oxidation. PAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the oxidation of N1-acetylated polyamines. The pH-dependence of the kcat/Kamine indicates that the monoprotonated form of the substrate is required for catalysis, with the N4 nitrogen next to the site of CH bond cleavage being unprotonated. Stopped-flow spectroscopy shows that the pH-dependence of the rate constant for flavin reduction, kred, displays a pKa of 7.3 with a decrease in activity at acidic pH. This is consistent with an uncharged nitrogen being required for catalysis. Mutating Lys315 to methionine has no effect on the kcat/Kamine-pH profile with the substrate spermine, and the kred value only shows a 1.5-fold decrease with respect to wild-type PAO. The mutation results in a 30- fold decrease in kcat/KO2. Solvent isotope effects and proton inventories are consistent with Lys315 accepting a proton from a water molecule hydrogen-bonded to the flavin N5 during flavin oxidation. Steady-state and transient kinetic studies of para-substituted N,N'-dibenzyl-1,4- diaminobutanes as substrates for PAO show that the kred values for each correlate with the van der Waals volume (VW) and the value. The coefficient for VW is the same at pH 8.6 and 6.6, whereas the p value increases from -0.59 at pH 8.6 to -0.09 at pH 6.6. These results are most consistent with a hydride transfer mechanism. The kinetics of oxidation of a peptide substrate by human lysine specific demethylase (LSD1) were also studied. The kcat/KM pH-profile is bell-shaped, indicating the need for one unprotonated nitrogen next to the site of CH bond cleavage and another protonated nitrogen. The kcat and kred values are equal, and identical isotope effects are observed on kred, kcat, and kcat/KM, indicating that CH bond cleavage is rate-limiting with this substrate.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains x, 121 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 84-118).

Fusarium graminearum causes Fusarium Head Blight, (one of) the most destructive cereal diseases in Canada. Yield loss, quality degradation and mycotoxin production make Fusarium a multifaceted threat. Regulated production of reactive oxygen species by Nox enzymes is indispensable for fungal pathogenesis. F. graminearum Nox mutant ∆noxAB produced equivalent mycotoxin but caused reduced virulence than wild-type. We hypothesized that Nox mediated redox signaling may participate in F. graminearum pathogenicity. Two-DE and gel-free biotin affinity chromatography, followed by LC-MS/MS analysis were employed for a comparative redox-proteomics analysis between wild-type and ∆noxAB to identify proteins oxidized by Nox activity. Total 35 proteins, 10 by 2-DE and 29 by gel-free system, were identified. 34% proteins participated in fungal metabolism, 20% in electron transfer reactions and 9% were anti-oxidant proteins. The findings suggested that Nox mediated thiol-disulfide exchange in proteins provide a switch for redox-dependent regulation of metabolic and developmental processes during induction of FHB.

Fusarium graminearum causes Fusarium Head Blight, (one of) the most destructive cereal diseases in Canada. Yield loss, quality degradation and mycotoxin production make Fusarium a multifaceted threat. Regulated production of reactive oxygen species by Nox enzymes is indispensable for fungal pathogenesis. F. graminearum Nox mutant ∆noxAB produced equivalent mycotoxin but caused reduced virulence than wild-type. We hypothesized that Nox mediated redox signaling may participate in F. graminearum pathogenicity. Two-DE and gel-free biotin affinity chromatography, followed by LC-MS/MS analysis were employed for a comparative redox-proteomics analysis between wild-type and ∆noxAB to identify proteins oxidized by Nox activity. Total 35 proteins, 10 by 2-DE and 29 by gel-free system, were identified. 34% proteins participated in fungal metabolism, 20% in electron transfer reactions and 9% were anti-oxidant proteins. The findings suggested that Nox mediated thiol-disulfide exchange in proteins provide a switch for redox-dependent regulation of metabolic and developmental processes during induction of FHB.

Atherosclerosis occurs preferentially at branches and curves in arteries exposed to disturbed flow while sparing straight portions of arteries exposed to undisturbed flow. In vivo and in vitro studies have implicated NADPH oxidases in atherosclerosis and hypertension. Shear stress can induce reactive oxygen species production in endothelial cells from a variety of sources, including NADPH oxidases. Here, we examined the hypothesis that unidirectional laminar shear (LS) and oscillatory shear (OS) would differentially regulate gene expression profiles in NADPH oxidase-dependent and -independent manners, and that these genes would provide novel molecular targets in understanding endothelial cell biology and vascular disease. The p47phox subunit of the NADPH oxidase can be an important regulator of certain Nox isoforms, including Nox1 and Nox2 which may be responsible for shear-induced superoxide production. In order to isolate p47phox-dependent shear responses, we took advantage of the p47phox-/- transgenic mouse model which lacks a functional p47phox subunit. We developed a method to isolate murine aortic endothelial cells using an enzymatic digestion technique. These cells expressed characteristic endothelial markers, including VE-cadherin, PECAM1, and eNOS, and aligned in the direction of flow. We successfully isolated primary murine aortic endothelial cells from both wild-type C57BL/6 mice (MAE-WT) and p47phox-/- mice (MAE-p47). Furthermore, we established an immortalized cell line from each of these cell types, iMAE-WT and iMAE-p47. We carried out microarray studies using Affymetrix Mouse Genome 430 2.0 Arrays (39,000+ transcripts) on MAE-WT and MAE-p47 that were exposed to atheroprotective LS or atherogenic OS for 24 hours. In comparison to LS, OS significantly changed the expression of 187 and 298 genes in MAE-WT and MAE-p47, respectively. Of those, 23 genes showed similar gene expression patterns in both cell types while 462 genes showed different gene expression patterns in the two cell types, demonstrating a considerable role for p47phox-based NADPH oxidases in shear-dependent gene expression. Changes in expression of several genes, including Kruppel-like factor 2 (Klf2), endothelial nitric oxide synthase (eNOS), angiopoietin 2 (Ang2), junctional adhesion molecule 2 (Jam2), bone morphogenic receptor type II (Bmpr2), and bone morphogenic protein 4 (Bmp4) were confirmed by quantitative PCR and/or immunoblotting using both primary cells and immortalized cells. Of these genes, our data suggest that Jam2, Bmpr2, and Bmp4 may be shear-sensitive in a p47phox-dependent manner. Taken together, our studies have identified a set of shear- and p47phox-sensitive genes, including unexpected and novel targets, which may play critical roles in vascular cell biology and pathobiology.

Several members of the lysyl oxidase family of copper-dependent amine oxidases have been implicated in tumor development. The Iysyl oxidase (LOX) and LOX-like 2 (LOXL2) genes have been mapped to chromosomal regions affected by loss of heterozygosity (LOH) in several cancers, including those of the colon and esophagus. Indeed, there have been numerous reports of reduced LOX and a few reports of reduced LOXL2 expression in various cancers. Identification of microsatellite markers within the LOX locus and the LOXL2 gene allowed for evaluation ofthe status of these gene alleles in colon and esophageal tumors. There was significant LOH of the LOX locus in colon tumors that was accompanied by reduced mRNA expression and a spectrum of alterations and mutations affecting the LOX gene. This study demonstrated, for the first time, that genetic events, namely LOH, deletions and mutations ofthe LOX gene, were responsible, at least partly, for the reduction of LOX gene expression. There was also significant LOH of the LOXL2 gene in both colon and esophageal tumors. However, instead of a reduction of LOXL2 expression, there was increased expression that correlated with less differentiated tumors and absent elastosis, both indicators of poor prognosis. Further studies indicated that both LOX and LOXL2 are absent in non-invasive tumor cell lines but re-expressed in invasive cell lines, likely as part of the thelial-mesenchymal transition that occurs in the last steps of tumorigenesis to facilitate metastasis. The results presented and research strategy outlined in this dissertation will define the importance of LOXL2 amine oxidase activity and protein interactions in the critical but poorly understood process oftumor cell migration and invasion.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (doctoral)--Universität Bern, 1913. / Includes bibliographical references.

Made available in DSpace on 2014-10-09T12:43:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:42Z (GMT). No. of bitstreams: 1 06648.pdf: 3159375 bytes, checksum: b1ad256abf7e9f79cfed807e33c3ce91 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:43:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:42Z (GMT). No. of bitstreams: 1 06648.pdf: 3159375 bytes, checksum: b1ad256abf7e9f79cfed807e33c3ce91 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP